This invention relates to treatment of HIV.
One of the classic markers of full-blown AIDS resulting from long-term infection with HIV-1 is a severe depletion of CD4+ T-cells, which are a key component of the immune system. Attempts have been made to increase the CD4+ counts of AIDS patients, and some of these efforts, notably treatment with HIV protease inhibitors, have met with considerable success. Other approaches, e.g., stimulation of the immune response by vaccination with viral peptides, have been less successful. The reasons for CD4+ depletion in AIDS, and resistance of CD4+ cells to stimulation by some therapies, are not fully understood.
The invention provides a new and highly advantageous method of potentiating the immune response in HIV-infected patients, employing extremely low concentrations of compounds which, at these concentrations, act as stimulatory, rather than inhibitory, molecules. The very low concentrations employed according to the invention allow treatment with minimal side effects and toxicity. The specificity of the treatments according to the invention also helps avoid these adverse effects, which are seen, for example, in treatment with immune stimulatory compounds such as Interleukin-2.
The invention involves the unexpected finding that certain compounds, which at relatively low concentrations (e.g. 10xe2x88x924-10xe2x88x926) are cytotoxic to T-cells, nevertheless have immune stimulatory properties at extremely low concentrations (e.g. 10xe2x88x9210-1014). Even more surprising, this stimulation occurs even though the HIV-infected patient""s T-cells are otherwise unable to respond to T-cell proliferation-inducing stimuli. The effects of these low concentrations are paraxodical because T-cells from non-HIV-infected individuals, which respond to T-cell proliferation-inducing stimuli, do not seem to respond in the same fashion as T-cells from an HIV-infected patient when treated with the extremely low concentrations of compounds according to the invention.
The invention thus provides a method for treating the T-cells of a human subject infected with human immunodeficiency virus. The subject""s T-cells are contacted with a molecule that inhibits CD26 and that stimulates immune function of the T-cells in an amount effective to stimulate immune function of the T-cells, said amount being below a concentration which causes detectable cytotoxicity of the T-cells. In one embodiment, the molecule stimulates proliferation of T-cells at the effective concentration. In other embodiments, the molecule stimulates the production of cytokines or increases cytotoxic T-lymphocyte or antibody activity.
The T-cells can be contacted in vitro or in vivo. In certain embodiments, the effective amount is below 10xe2x88x928M and may be between 10xe2x88x9210 and 10xe2x88x9216 M. Molarity is measured as a function of final concentration in vitro and as a function of blood concentration in vivo.
According to another aspect of the invention, the molecule can be administered in conjunction with a different therapeutic agent that increases the CD4+ count of HIV-infected subjects. In this manner, treatment with effective amounts of the molecule according to the invention can enhance the therapeutic effect of other AIDS drugs. Specifically contemplated is use of the molecules according to the invention with non CD26 protease inhibitors. It has been determined that the effects of the treatments according to the invention are particularly good in patients whose CD4+ count is above about 400. In one embodiment, subjects may be treated with therapeutic agents or regimens to increase T-cell count to above 400 where the count initially is below 400. The subjects then are believed to be better candidates for treatment according to the invention. Thus, the invention contemplates the use of the molecules according to the invention to provide optimal combination AIDS therapies. Particularly contemplated for use in conjunction with the molecules of the invention are therapeutics which inhibit HIV replication by, for example, inhibiting reverse transcriptase or by inhibiting HIV protease activity. Exemplary therapeutics include the antiretroviral drugs: AZT (3xe2x80x2 azido-2xe2x80x2,3xe2x80x2 dideoxythymidine), ddI (2xe2x80x2,3xe2x80x2 dideoxyinosine), (2xe2x80x2,3xe2x80x2 dideoxycytidine), ddt (2xe2x80x2,3xe2x80x2 didehydro-3xe2x80x2-deoxythymidine) and other nucleoside and non-nucleoside reverse transcriptase inhibitors and HIV-1 protease inhibitors: Indinavir, Ritonavir, Saquinavir. Combination therapies include the administration of one or more of these or other antiretroviral therapeutics, alone or together with therapeutics that are intended to treat the secondary infections associated with HIV and/or cytokines (e.g., GM-CSF, G-CSF, interferons, interleukins)
According to another aspect of the invention, the molecules of the invention are contacted with T-cells of a human subject infected with HIV, which T-cells are unable, prior to treatment according to the invention to respond normally to T-cell proliferation-inducing stimuli. In one particularly important embodiment, the molecules of the invention are administered in conjunction with an antigen. Ordinarily, such HIV-infected patients do not respond well to antigens because of an HIV-induced defect in the T-cell stimulation pathway. Use of the molecules according to the invention as adjuvants can render such T-cells responsive to stimulation or vaccination with antigens. The invention permits immunization of HIV-infected patients with antigens characteristic of HIV, antigens characteristic of other pathogens, antigens characteristic of cancer cells and the like. Peptide antigens in particular are contemplated.
Compounds useful in the invention inhibit CD26 and stimulate proliferation of T-cells of HIV-infected subjects in the assays and at the concentrations described below. CD26 is also referred to as xe2x80x9cdipeptidyl-aminopeptidase type-IVxe2x80x9d or xe2x80x9cDP-IVxe2x80x9d. CD26 is a post-prolyl cleaving enzyme with a specificity for removing Xaa-Pro (where Xaa represents any amino acid) dipeptides from the amino terminus of a polypeptide substrate.
Peptides which reportedly have demonstrated utility for inhibiting post-prolyl cleaving enzymes and which, if coupled to a reactive group, form a covalent complex with a functional group in the reactive site of a post-prolyl cleaving enzyme are described in U.S. Pat. No. 4,935,493, xe2x80x9cProtease Inhibitorsxe2x80x9d, issued to Bachovchin et al. (xe2x80x9cBachovchin ""493xe2x80x9d); U.S. Pat. No. 5,462,928, xe2x80x9cInhibitors of Dipeptidyl-aminopeptidase Type IVxe2x80x9d, issued to Bachovchin et al. (xe2x80x9cBachovchin ""928xe2x80x9d); U.S. Pat. No. 5,543,396, xe2x80x9cProline Phosphonate Derivativesxe2x80x9d, issued to Powers et al., (xe2x80x9cPowers ""396xe2x80x9d); U.S. Pat. No. 5,296,604, xe2x80x9cProline Derivatives and Compositions for Their Use as Inhibitors of HIV Proteasexe2x80x9d, issued to Hanko et al., (xe2x80x9cHanko ""604xe2x80x9d); PCT/US92/09845, xe2x80x9cMethod for Making a Prolineboronate Esterxe2x80x9d, and its U.S. priority applications (U.S. Ser. Nos. 07/796,148 and 07/936,198), Applicant Boehringer Ingelheim Pharmaceuticals, Inc. (xe2x80x9cBoehringerxe2x80x9d). Representative structures of the transition-state analog-based inhibitors Xaa-Boo-Pro, include Lys-boroPro, Pro-boroPro, Val-boroPro and Ala-boroPro in which xe2x80x9cboroProxe2x80x9d refers to the analog of proline in which the carboxylate group (COOH) is replaced with a boronyl group (B) (OH2).
In general, the molecules useful according to the invention can have a first targeting moiety for binding a post-prolyl cleaving enzyme such as CD26 covalently coupled to a first reactive group. As used herein, a reactive group is capable of reacting with a functional group in a post-prolyl cleaving enzyme such as CD26. By reacting, it is meant that the reactive group forms a bond with a functional group of a post-prolyl cleaving enzyme such as CD26. Reactive groups that are embraced within the invention include the reactive groups referred to as group xe2x80x9cTxe2x80x9d in U.S. Pat. No. 4,935,493, xe2x80x9cProtease Inhibitorsxe2x80x9d, issued to Bachovchin, et al. These include boronate groups, phosphonate groups, and fluoroalkylketone groups. In general, it is preferred that the linkage between the carboxyl terminus of preferred peptide targeting moieties and the reactive group be in an L configuration. It is preferred that the reactive group form a covalent bond with a functional group of the active site of a reactive center of CD26; however, there is no requirement for covalent bond formation in order to form a complex between the CD26 binding molecule and CD26.
The reactive groups that are fluoroalkylketone groups have the formula: 
where G is either H, F or an alkyl group containing 1 to about 20 carbon atoms and optional heteroatoms which can be N, S, or O. Additional exemplary proline phosphonate derivatives which contain a perfluoroalkyl group, a phenyl group or a substituted phenyl group and which can be used in accordance with the methods of the invention are those described in U.S. Pat. No. 5,543,396 (Powers ""396).
The reactive groups that are phosphonate groups have the formula: 
where each J, independently, is O-alkyl, N-alkyl, or alkyl (each containing about 1-20 carbon atoms) and, optionally, heteroatoms which can be n=N, S, or O. Other ketoamides, ketoacids and ketoesters that are useful reactive groups for reacting with the reactive center of a protease are described in PCT/US91/09801, xe2x80x9cPeptides, Ketoamides, Ketoacids, and Ketoestersxe2x80x9d, Applicant: Georgia Tech Research Corp. (xe2x80x9cGA Techxe2x80x9d) which claims priority to U.S. Pat. No. 635,287, filed Dec. 28, 1990.
In certain embodiments, the reactive groups are selected from the groups having the formulas 
an alphaketo amide; 
where R is an alkyl, or aryl group and may be substituted or unsubstituted, an alphaketo ester; and 
an alphaketo acid.
The preferred compounds have targeting moieties that are peptides which mimic the substrate binding site of DP-IV. Peptide analogs and nonpeptides or peptidomimetics also can be used as targeting moieties. Such molecules can be rationally designed based upon the known sequence of substrates of DP-IV or can be identified using combinatorial chemistry and screening assays such as are described below.
The development of phage display libraries and chemical combinatorial libraries permits the selection of synthetic compounds which mimic the substrate binding site of a protease such as CD26. Such libraries can be screened to identify non-naturally occurring putative targeting moieties by assaying protease cleavage activity in the presence and absence of the putative phage display library molecule or combinatorial library molecule and determining whether the molecule inhibits cleavage by the protease of its natural substrate or of a substrate analog (e.g., a chromophoric substrate analog which is easily detectable in a spectrophotometric assay). Those phage library and/or combinatorial library molecules which exhibit inhibition of the protease then can be covalently coupled to the reactive groups R disclosed herein and again tested to determine whether these novel molecules selectively bind to the protease (e.g., by repeating the above-noted screening assay). In this manner, a simple, high-through-put screening assay is provided for identifying non-naturally occurring targeting moieties of the invention.
Compounds useful in the invention include, but are not limited to, compounds that inhibit CD26 and are embraced by the following formula PR, wherein P represents a targeting moiety that binds to CD26 and R represents a reactive group that reacts with a functional group in CD26, preferably a reactive center of CD26. P can be any molecule that binds CD26 including CD26 binding molecules embraced by the formula: Dxe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94A4, wherein D is independently selected from the group consisting of NH and NH2, wherein N represents any isotope of nitrogen, herein H represents any isotope of hydrogen; xe2x80x9cxe2x80x94xe2x80x9d, independently, is selected from the group consisting of a single bond and a double bond; A1 is selected from the group consisting of a C, a CX and an N, wherein C represents any isotope of carbon, X represents any atom that forms a single band with carbon; each A2, A3, and A4, independently, is selected from the group consisting of a CX moiety, a CXZ moiety, a CZ moiety, an NX moiety, and an O, wherein X and Z, independently are selected from the group consisting of any atom that forms a single bond and any atom that forms a double bond with C or N and wherein O represent any isotope of oxygen.
Compounds useful according to the invention also include the following of Group I or Group II. Group I has the structure: 
where H represents a hydrogen: C represents a carbon; O represents an oxygen; N represents a nitrogen; each R, independently, is chosen from the group consisting of the R groups of an amino acid, including proline; each broken line, independently, represents a bond to an H or a bond to one R group, and each Hxe2x80x2 represents that bond or a hydrogen; and p is an integer between 0 and 4 inclusive. Alternatively, Group I has the structure: 
where n is between 0 and 3 inclusive, each G2 and G3 independently is H or C1-3 (one to three carbon atoms) alkyl, G1 is NH3 (H3 represents three hydrogens), 
(H2 represents two hydrogens), or NG4, where G4 is 
where G5 and G6 can be NH, H, or C1-3 alkyl or alkenyl with one or more carbons substituted with a nitrogen. G1 bears a charged, and G1 and Group II do not form a covalently bonded ring structure at pH 7.0. Group I may also have the structure: 
where one or two of the a, b, c, d, e, and f group is N. and the rest are C, and each S1-S6 independently is H or C1-C3 alkyl. Group I may also include a five membered unsaturated ring having two nitrogen atoms, e.g., an imidazole ring. Group II has the structure: 
where T is a group of the formula: 
where each D1 and D2, independently, is a hydroxyl group or a group which is capable of being hydrolysed to a hydroxyl group in aqueous solution at physiological pH; a group of the formula: 
where G is either H, fluorine (F) or an alkyl group containing 1 to 20 carbon atoms and optional heteroatoms which can be N, S (sulfur) or O; or a phosphonate group of the formula: 
where each J, independently, is O-alkyl, N-alkyl, or alkyl. Each O-alkyl, N-alkyl or alkyl includes 1-20 carbon atoms and, optionally, heteroatoms which can be N, S, or O. T is generally able to form-a complex with the catalytic site of a DP-IV. Y is 
and each R1, R2, R3, R4, R5, R6, R7, and R8, separately is a group which does not significantly interfere with site specific recognition of the inhibitory compound by DP-IV, and allows a complex to be formed with DP-IV.
In preferred embodiments, T is a boronate group, a phosphonate group or a trifluoroalkyl ketone group; each R1-R8 is H; each R1 and R2 is H, and each Y is the CH2xe2x80x94CH2; each R is independently chosen from the R group of proline and alanine; the inhibitory compound has a binding or dissociation constant to DP-IV of at least 10xe2x88x929M, 10xe2x88x928M or even 10xe2x88x927M; and each D1 and D2 is, independently, F or D1 and D2 together are a ring containing 1 to 20 carbon atoms, and optionally heteroatoms which can be N, S, or O. These compounds are described in U.S. Pat. No. 5,462,928, hereby incorporated by reference.
More preferably, the compound used at low concentration according to the invention has the formula 
wherein m is an integer between 0 and 10, inclusive; A and A1 are amino acid residues such that the A in each repeating bracketed unit can be a different amino acid residue; the bonds between A and N. A1 and C, and between A1 and N are peptide bonds; and each X1 and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group at physiological pH. These compounds are also described in U.S. Pat. No. 5,462,928.
The most preferred compounds are of the formula 
where each D1 and D2, independently, is a hydroxyl group or a group which is capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH;
and wherein X is a targeting moiety that mimics the site of a substrate recognized and cleaved by CD26.
Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.